Project Summary/ Abstract Regular physical activity is associated with reduced incidence of chronic pain in epidemiological studies; yet, an acute bout of exercise can exacerbate pain in those with chronic pain. Analogously in animal models, we show a single bout of exercise enhances the nociceptive response to muscle insult in physically inactive mice, and that regular physical activity prevents the development of chronic muscle hyperalgesia. The mechanisms driving this apparent dichotomy in pain response to physical activity are poorly understood. We propose this dichotomy may in large part be explained by the plasticity of local muscle macrophages. Macrophages release inflammatory or anti-inflammatory cytokines depending on two relevant phenotypes: classically-activated (M1) macrophages release inflammatory cytokines and regulatory (M2) macrophages release anti-inflammatory cytokines. The relative proportion of macrophage phenotype dictates the immune response, and we propose that regular physical activity shifts the balance between M1 and M2 macrophages to result in greater release of anti-inflammatory cytokines. Fatigue metabolites, adenosine triphosphate (ATP) and protons, produce pain in humans and hyperalgesia in inactive animals, and activate surface receptors, P2X7 and ASIC3, on macrophages. Our preliminary data support a role for macrophages in both activity-induced pain and activity- induced analgesia and show a greater proportion of M2s after regular exercise. Specific Aim 1 will investigate the role of macrophages in activity-induced hyperalgesia in physically inactive mice. We hypothesize that activation of P2X7 or ASIC3 on muscle macrophages releases IL-1? in physically inactive animals to produce hyperalgesia. Specific Aim 2 will characterize the role of macrophages in prevention of chronic muscle pain by regular physical activity. We hypothesize that activation of P2X7 or ASCI3 on muscle macrophages releases IL-10 in physically active animals to produce analgesia. Specific Aim 3 will investigate if ATP, protons, or their combination produces a phenotypic switch in cultured macrophages from M1 to M2. We hypothesize that the combination of ATP and protons is necessary to induce the phenotypic switch from M1 to M2 macrophages. These studies will examine the interactions between muscle, macrophages, and nociceptors and thus will be the first to determine the role of the innate immune system in activity-induced hyperalgesia and analgesia. Understanding these mechanisms is critically important to understanding the development and prevention of chronic pain, and the consequences of physical activity in individuals with pain. Treatments aimed at reducing pain during an initial exercise program could lead to better adherence in maintaining regular physical activity for people with chronic pain. Further determining factors activated by regular physical activity, which activates endogenous resolution mechanisms, is a critical component to preventing the transition from acute to chronic pain.